Kernicterus is a cause of newborn brain disorder. In full-term infants (mature infants), nearly all cases of kernicterus are preventable by early detection and prompt treatment because of the progress in perinatal medical care. As for preterm infants, on the other hand, the number of cases of kernicterus diagnosis has been recently increasing due to, for example, an increase in the survival rate of extremely preterm infants. This trend is a great concern in the field of pediatric and newborn medical care and needs to be addressed urgently.
It is well known that kernicterus is caused by jaundice developed in the newborn period, more specifically, developed by serum bilirubin. Serum bilirubin, which is often called total bilirubin (TB), is a yellow substance that contains unconjugated bilirubin (indirect bilirubin, iDB) and conjugated bilirubin (direct bilirubin, DB). Unconjugated bilirubin has neurotoxicity. A type of unconjugated bilirubin not bound to albumin, namely unbound bilirubin (UB), has a low molecular weight and therefore can easily cross the blood-brain barrier and deposit in the brain. This phenomenon is a significant cause of neurotoxicity.
Techniques for unbound bilirubin measurement have been studied since the 1960s. Among these efforts, inventors of the present invention have reported the glucose oxidase-peroxidase (GOD-POD) method (see Non-patent Document 1: Clin Chim Acta. 1977 Sep. 1; 79(2): 411-7). The inventors have also developed an automatic measurement apparatus (UB-Analyzer manufactured by Arrows) that is based on the GOD-POD method (see Non-patent Document 2: Kobe J Med Sci. 1982 April: 28(2): 91-104). This automatic measurement apparatus is the only clinical apparatus for measuring unbound bilirubin, and is approved by U.S. Food and Drug Administration (FDA) and Japan's Ministry of Health, Labour and Welfare.
The GOD-POD method uses glucose and glucose oxidase to generate hydrogen peroxide and then makes peroxidase act on the hydrogen peroxide to induce oxidative decomposition of bilirubin. In this method, albumin-unbound bilirubin is oxidatively decomposed and readily converted into a colorless substance, whereas albumin-bound bilirubin tends not to be oxidatively decomposed. Therefore, unbound bilirubin concentrations are calculated from the initial rate of the oxidative decomposition. More specifically, unbound bilirubin concentrations are determined by colorimetrically monitoring the decreasing rate of bilirubin pigments.
The criteria for jaundice treatment published from Department of Pediatrics, Kobe University Graduate School of Medicine define the suitable range of concentrations of both serum total bilirubin and unbound bilirubin for indication of phototherapy and exchange transfusion (see Non-patent Document 3: Edited by Department of Pediatrics, Kobe University Graduate School of Medicine, Management in preterm infants and newborns. Tokyo. Nihon Shoni Iji Shuppan-sha, 1991). The wide use of this criteria has greatly contributed to reducing the incidence of kernicterus in mature infants. As for the application to preterm infants, however, some has pointed out that the criteria can lead to overtreatment and therefore the rate of compliance has been decreasing.
There is another set of criteria for jaundice treatment, which exclusively relies on serum total bilirubin concentrations for indication of phototherapy and exchange transfusion (Non-patent Document 4: Yasuhiro Kawase. Neonatal hyperbilirubinemia. Toru Yamaguchi, Mitsuo Kitahara, Tsuguya Fukui, Omnibus, Today's Therapy 2006, Igaku-Shoin Ltd., Tokyo, 2006, pp 941-942).
There is a study that a gene from Japanese eel muscle has been isolated, and the gene codes for a protein that emits green fluorescence. The researchers of this study have found that its gene product, UnaG, specifically binds to unconjugated bilirubin and emits an intense green fluorescence, and that the UnaG-bound bilirubin acts as a fluorescent chromophore (see Non-patent Document 5: Cell. 2013 Jun. 20; 153(7): 1602-11, and Patent Document 1: WO 2014/133158 A).